Colon transcriptome is modified by a dietary pattern/atorvastatin interaction in the Ossabaw pig

 Optimizing diet quality in conjunction with statin therapy is currently the most common approach for coronary artery disease (CAD) risk management

• Although effects on the cardiovascular system have been extensively investigated, little is known about the effect of these interventions in the colon and subsequent associations with CAD progression.
• In this study, Ossabaw pigs were randomly allocated to receive, for a six-month period, isocaloric amounts of either a heart healthy-type diet (HHD; high in unrefined carbohydrate, unsaturated fat, fiber, supplemented with fish oil, and low in cholesterol) or a Western type diet without or with atorvastatin therapy.

Cardiovascular disease (CVD) is the leading cause of death globally

• Approximately one-third of US adult deaths are attributable to CVD.
• Evidence-based lifestyle recommendations for the prevention and management of CAD include adopting a heart-healthy dietary pattern, defined by the American Heart Association (AHA) and American College of Cardiology (ACC) as rich in fruits, vegetables, whole grains, healthy proteins, nuts, seeds and legumes, while limiting intake of sodium, saturated fat, processed meats and sugar-sweetened beverages.

Study design and animals

• Thirty-two 5-8 week old pigs (16 boars+16 gilts) were randomly allocated to one of four groups using a 2 × 2 factorial design: WD−S, WD+S, HHD−S and HHD+S
• An equal number of boars and gilts was allocated in each group.
• After a one-month acclimation period the pigs were gradually shifted to their respective experimental diets for an addition 6 months, with incremental increases in energy to meet growth requirements.

Diets and atorvastatin therapy

• Designed to be isocaloric and reflect typical human Western and heart healthy dietary patterns
• 47% of energy (E) as carbohydrate, 38% of E as fat, and 15% E as protein
• The diets differed in the types of carbohydrate and fat, quantity of cholesterol and fiber, and fish oil supplementation.

Sample collection

• At the end of the intervention period, pigs were euthanized by an intravenous injection of Euthasol (50 mg sodium pentobarbital/kg body weight)
• Proximal colon segments (2 cm in length) were harvested from an anatomically similar region, cleaned and rinsed with PBS, flash-frozen in liquid nitrogen, and stored at −80°C.

Sample processing

• Serum cardiometabolic risk factors, including LDL cholesterol, high-density lipoprotein (HDL) cholesterol, triglyceride, tumor necrosis factor-alpha (TNF-α), and high-sensitivity C-reactive protein (hsCRP) concentrations, were measured and reported.

Isolation of colonic mucosa and RNA extraction

• Frozen colon segments were treated with prechilled RNAlater-ICE (Invitrogen, Carlsbad, CA) at −20°C for 24 hours to preserve RNA quality and prepare samples for further dissection.
• Colon segments were opened longitudinally, and the mucosal layer was cleanly separated from the submucosal layer using scalpel and tweezers.

RNA sequencing

• The sample libraries were prepared using Illumina TruSeq RNA Sample Preparation Kit v2.5 (Illumina, San Diego, CA) and AMPure XP beads (Beckman Coulter, Hercules, CA).
• Libraries were quantified using a KAPA Library Quantification kit (KAPA Biosystems, Wilmington, MA) and Experion DNA 1K Analysis kit.
• Raw data in FASTQ format was trimmed for quality by CLC Bio Genomic Workbench.

Characterizing colonic mucosa cell types and sample homogeneity

• xCell tool was used to analyze the RNA sequencing data (reads per kilobase million [RPKM]) that predicted enrichment of various cell types within each colon sample.
• The epithelial cell enrichment data among the four groups was analyzed by one-way ANOVA (Prism 8, GraphPad Software, La Jolla, CA). No significant differences were identified, suggesting similar enrichment of colonic mucosa among groups.

Differential expression analysis of RNA-seq data and gene enrichment analysis

• Genes were considered differentially expressed based on a false discovery rate (FDR) ≤ 0.05 and absolute log fold change (logFC) ≥0.6 (absolute fold change ≥1.5).
• Fold change for genes was interpreted as diet effect (WD vs. HHD) and statin effect (+S vs. −S). An interaction of diet-statin with FDR<0.05 was considered significant.
• To further assess potential interactions by dietary patterns or atorvastatin therapy, analyses adopting an exact test model were conducted in edgeR [24].
• Comparison pairs included diet effect within statin groups (WD−S, and WD+S relative to HHD+S) and cholesterol effect within diet groups.

Analysis Match with public gene expression datasets

• To compare the derived biological interpretation of our dataset to other analyses, Analysis Match in IPA was used.
• The algorithm created a signature from the highest confidence predictions from our query analysis and compared it to the signatures of analyses generated from public gene Expression datasets curated by OmicSoft, ArrayExpress, etc.

Correlation analyses among gene expression and clinical traits

• To determine the association of gene expression in colonic mucosa with atherosclerotic lesion severity and cardiometabolic risk factors, pigs from all groups were pooled (n=29).
• The differentially expressed genes and genes involved in pathways altered by dietary patterns and/or atorvastatin therapy were included in this analysis.
• Spearman's correlation coefficients were calculated. An association was considered statistically significant when absolute correlation coefficient r≥0.4 with a P value ≤.05.

Sex difference

• A descriptive secondary analysis was performed in colonic mucosa to determine whether boars and gilts differentially respond to the interventions, using the methods described in the Section "Differential expression analysis of RNA-seq data and gene enrichment analysis."

Differential gene expression analysis

• Thirty-one differentially expressed genes with FDR≤0.05 and absolute logFC≥0.6 were identified in colonic mucosa attributable to dietary patterns, atorvastatin therapy, and/or their interaction (Table 1).
• The expression of 10 genes demonstrated a significant diet-statin interaction.

Gene enrichment analysis

• IPA was used to evaluate gene enrichment. Genes with absolute logFC≥0.6 were included to extend our ability to explore potential pathways and biological functions altered by dietary patterns and atorvastatin therapy.
• The trend of a diet-statin interaction was identified by IPA Comparison Analysis (Fig. 1).
• Results from the pathway analyses were similar regardless of the databased used.

Analysis Match with public gene expression datasets

• The IPA Analysis Match was conducted to further elucidate insights regarding how atorvastatin therapy affects colonic gene expression within different diet context.
• Results indicated that the colonic mucosa gene expression pattern of WD+S relative to WD-S fed pigs was similar to that of a microbiota dysbiosis phenotype relative to normal control (mouse colon, Z score=77.96% on predicted Upstream Regulators) and a ulcerative colitis phenotype compared to healthy control.

Differentially expressed genes

• The expression of ASS1, CD274, GBP2, and SLC6A9 in the colonic mucosa were negatively associated with serum hsCRP concentrations.
• CLEC4GC-type lectin domain family 4 member G (P value)
• ASS1argininosuccinate synthase
• CD274CD274 molecule
• Gbp2guanylate binding protein 20.9
• Analysis conducted independent of treatments (n=29)

Genes in pathways altered by dietary patterns

• Among genes expressed in "LXR/RXR Activation" pathway, MMP9 was positively associated with atherosclerotic lesion severity, serum LDL cholesterol, HDL cholesterol, and triglyceride concentrations.
• PTGS2 was negatively associated with cholesterol, TNF-α, and LYZ concentrations.
• PLA2G3 and IRAK3 expressed in both "Phospholipase" and "p38 MAPK Signaling" pathways were negatively related to cholesterol, triglyceride, and LDL cholesterol concentrations.

Genes in pathways altered by atorvastatin therapy

• Only the expression of CR2 gene in "PI3K Signaling in B Lymphocytes" was positively associated with atherosclerotic lesion severity (Table 5).
• The gene expression of CCR3, ICOS, CYBB, TNFSF11, ATF3, CD180 in various pathways were negatively associated with serum hsCRP concentrations.
• expression of IL10, PLA2G3, and LYZ in "Production of Nitric Oxide and Reactive Oxygen Species in Macrophages" pathway was negatively related to serum triglyceride concentrations.

Discussion Recent findings suggest there is an interplay between the gut and heart, referred to as the heart-gut axis, and that this relationship can be exploited for use as a therapeutic target for CAD risk reduction.

• Yet, despite the widespread use of statins as a therapy to lower CAD risk, little is known about the potential pleotropic effects of statin therapy in the colon or potential interactions with dietary modification.

Diet effects

• Compared to the HHD, the WD downregulated "p38 MAPK" and "TREM 1 Signaling" pathway in the colonic mucosa.
• Among the diet-altered pathways, the MMP9 gene expression in "LXR/RXR Activation" pathway was positively associated with atherosclerotic lesion severity, and serum LDL cholesterol and HDL cholesterol concentrations.

Statin effects

• The vast majority of the differentially expressed genes were attributable to atorvastatin therapy, and about one-third of the genes had a significant diet-statin interaction.
• In Ossabaw pigs fed the HHD, but not WD, in lowering inflammatory status in colonic mucosa
• Although none of the pathways assessed in the WD-fed pigs were significantly altered, functional annotation analysis suggested that the drug may have triggered colonic inflammation.

Diet-statin interaction

• Differential gene expression and pathway analyses identified this interaction
• About 1/3 of the differentially expressed genes showed significant interactions, and the main diet effect was only observed in the pigs not receiving atorvastatin.
• Reasons for these interactions may result from factors associated with changes in the gut microbiome.

Strengths and limitations

• The diets were formulated to mimic those habitually consumed by humans, intending to simulate two dietary patterns, which allow the study of diet from a holistic rather than individual food or nutrient perspective. The atorvastatin doses were chosen to mimic a dose typically prescribed for human.
• A limitation of this work is that RNA was isolated from mucosal tissue homogenates that contained multiple cell types, hence, high sampling heterogeneity may have resulted in contamination of RNA from neurons and myocytes.

Conclusion

• The data indicate that dietary patterns and atorvastatin therapy differentially altered the colonic gene expression phenotype, with diet-statin interactions in Ossabaw pigs.
• Interactions suggested a potential side-effect of atorvasastin therapy on colonic mucosa within the context of a WD, emphasizing the critical role of diet quality in modulating response.

Reference

10.1016/j.jnutbio.2020.108570